A Tyrosine Aminomutase from Rice (Oryza sativa) Isomerizes (S)-α- to (R)-ß-Tyrosine with Unique High Enantioselectivity and Retention of Configuration.

A recently discovered 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (OsTAM) from rice [Yan, J., et al. (2015) Plant Cell 27, 1265] converts (S)-α-tyrosine to a mixture of (R)- and (S)-ß-tyrosines, with high (94%) enantiomeric excess, which does not change with pH, like it does for two bacterial TAMs. The K(M) of 490 µM and the k(cat) of 0.005 s(-1) are similar for other TAM enzymes. OsTAM is unique and also catalyzes (R)-ß- from (S)-α-phenylalanine. OsTAM principally retains the configuration at the reactive C(α) and C(ß) centers during catalysis much like the phenylalanine aminomutase on the Taxol biosynthetic pathway in Taxus plants.

A Photopolymerized Hydrogel System with Dual Stiffness Gradients Reveals Distinct Actomyosin-Based Mechano-Responses in Fibroblast Durotaxis.

Durotaxis, migration of cells directed by a stiffness gradient, is critical in development and disease. To distinguish durotaxis-specific migration mechanisms from those on uniform substrate stiffnesses, we engineered an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with dual slopes (steep and shallow), adjacent to uniform stiffness (soft and stiff) regions. While fibroblasts rely on nonmuscle myosin II (NMII) activity and the LIM-domain protein Zyxin, ROCK and the Arp2/3 complex are surprisingly dispensable for durotaxis on either stiffness gradient. Additionally, loss of either actin-elongator Formin-like 3 (FMNL3) or actin-bundler fascin has little impact on durotactic response on stiffness gradients. However, lack of Arp2/3 activity results in a filopodia-based durotactic migration that is equally as efficient as that of lamellipodia-based durotactic migration. Importantly, we uncover essential and specific roles for FMNL3 and fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis response to the stiffness gradients. Together, our tunable all-in-one hydrogel system serves to identify both conserved as well as distinct molecular mechanisms that underlie mechano-responses of cells experiencing altered slopes of stiffness gradients.

AGC kinase inhibitors regulate STING signaling through SGK-dependent and SGK-independent mechanisms.

Type 1 IFN expression is critical in the innate immune response, but aberrant expression is associated with autoimmunity and cancer. Here, we identify N-[4-(1H46 pyrazolo[3,4-b] pyrazin-6-yl)-phenyl]-sulfonamide (Sanofi-14h), a compound with preference for inhibition of the AGC family kinase SGK3, as an inhibitor of Ifnb1 gene expression in response to STING stimulation of macrophages. Sanofi-14h abrogated SGK activity and also impaired activation of the critical TBK1/IRF3 pathway downstream of STING activation, blocking interaction of STING with TBK1. Deletion of SGK1/3 in a macrophage cell line did not block TBK1/IRF3 activation but decreased expression of transcription factors, such as IRF7 and STAT1, required for the innate immune response. Other AGC kinase inhibitors blocked TBK1 and IRF3 activation suggesting common action on a critical regulatory node in the STING pathway. These studies reveal both SGK-dependent and SGK-independent mechanisms in the innate immune response and indicate an approach to block aberrant Ifnb1 expression.

Coro1B and Coro1C regulate lamellipodia dynamics and cell motility by tuning branched actin turnover.

Actin filament dynamics must be precisely controlled in cells to execute behaviors such as vesicular trafficking, cytokinesis, and migration. Coronins are conserved actin-binding proteins that regulate several actin-dependent subcellular processes. Here, we describe a new conditional knockout cell line for two ubiquitous coronins, Coro1B and Coro1C. These coronins, which strongly co-localize with Arp2/3-branched actin, require Arp2/3 activity for proper subcellular localization. Coronin null cells have altered lamellipodial protrusion dynamics due to increased branched actin density and reduced actin turnover within lamellipodia, leading to defective haptotaxis. Surprisingly, excessive cofilin accumulates in coronin null lamellipodia, a result that is inconsistent with the current models of coronin-cofilin functional interaction. However, consistent with coronins playing a pro-cofilin role, coronin null cells have increased F-actin levels. Lastly, we demonstrate that the loss of coronins increases accompanied by an increase in cellular contractility. Together, our observations reveal that coronins are critical for proper turnover of branched actin networks and that decreased actin turnover leads to increased cellular contractility.